Detecting Hematopoietic Stem Cell Proliferation Using BrdU Incorporation

Matatall, Katie A.; Kadmon, Claudine S.; King, Katherine Y.

doi:10.1007/978-1-4939-7371-2_7

Cited by 28 publications

(18 citation statements)

References 31 publications

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“…Cell proliferation within the thyroid tumour tissue was investigated by IHC. Two markers were used; Ki67, a nuclear protein expressed during all active phases of the cell cycle (non-senescent cells) and BrdU which is incorporated during s-phase of the cell cycle [ 24 ]. This analysis demonstrated that proliferating cells were detectable in both freshly resected tissue and post-culture tissue.…”

Section: Resultsmentioning

confidence: 99%

A novel microfluidic device capable of maintaining functional thyroid carcinoma specimens ex vivo provides a new drug screening platform

et al. 2019

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BackgroundThough the management of malignancies has improved vastly in recent years, many treatment options lack the desired efficacy and fail to adequately augment patient morbidity and mortality. It is increasingly clear that patient response to therapy is unique to each individual, necessitating personalised, or ‘precision’ medical care. This demand extends to thyroid cancer; ~ 10% patients fail to respond to radioiodine treatment due to loss of phenotypic differentiation, exposing the patient to unnecessary ionising radiation, as well as delaying treatment with alternative therapies.MethodsHuman thyroid tissue (n = 23, malignant and benign) was live-sliced (5 mm diameter × 350-500 μm thickness) then analysed or incorporated into a microfluidic culture device for 96 h (37 °C). Successful maintenance of tissue was verified by histological (H&E), flow cytometric propidium iodide or trypan blue uptake, immunohistochemical (Ki67 detection/ BrdU incorporation) and functional analysis (thyroxine [T4] output) in addition to analysis of culture effluent for the cell death markers lactate dehydrogenase (LDH) and dead-cell protease (DCP). Apoptosis was investigated by Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL). Differentiation was assessed by evaluation of thyroid transcription factor (TTF1) and sodium iodide symporter (NIS) expression (western blotting).ResultsMaintenance of gross tissue architecture was observed. Analysis of dissociated primary thyroid cells using flow cytometry both prior to and post culture demonstrated no significant change in the proportion of viable cells. LDH and DCP release from on-chip thyroid tissue indicated that after an initial raised level of release, signifying cellular damage, detectable levels dropped markedly. A significant increase in apoptosis (p < 0.01) was observed after tissue was perfused with etoposide and JNK inhibitor, but not in control tissue incubated for the same time period. No significant difference in Ki-67 positivity or TTF1/NIS expression was detected between fresh and post-culture thyroid tissue samples, moreover BrdU positive nuclei indicated on-chip cellular proliferation. Cultured thyroid explants were functionally viable as determined by production of T4 throughout the culture period.ConclusionsThe described microfluidic platform can maintain the viability of thyroid tissue slices ex vivo for a minimum of four days, providing a platform for the assessment of thyroid tissue radioiodine sensitivity/adjuvant therapies in real time.

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Section: Resultsmentioning

confidence: 99%

A novel microfluidic device capable of maintaining functional thyroid carcinoma specimens ex vivo provides a new drug screening platform

et al. 2019

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“…Other markers include the DNA intercalating agents BrdU and EdU, which are absent or poorly retained in quiescent cells. However, both BrdU and EdU are unsuitable for downstream and lengthy analysis because they harm cells and induce mutations [ 81 ]. Fluorescent dyes are categorized according to their binding affinity to: (a) nucleic acids, (b) cytoplasm, (c) membrane, and d) cell cycle-related proteins.…”

Section: Measurement Of Quiescent Cancer Cellsmentioning

confidence: 99%

Towards a Framework for Better Understanding of Quiescent Cancer Cells

Nabil

Song

et al. 2021

Cells

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Quiescent cancer cells (QCCs) are cancer cells that are reversibly suspended in G0 phase with the ability to re-enter the cell cycle and initiate tumor growth, and, ultimately, cancer recurrence and metastasis. QCCs are also therapeutically challenging due to their resistance to most conventional cancer treatments that selectively act on proliferating cells. Considering the significant impact of QCCs on cancer progression and treatment, better understanding of appropriate experimental models, and the evaluation of QCCs are key questions in the field that have direct influence on potential pharmacological interventions. Here, this review focuses on existing and emerging preclinical models and detection methods for QCCs and discusses their respective features and scope for application. By providing a framework for selecting appropriate experimental models and investigative methods, the identification of the key players that regulate the survival and activation of QCCs and the development of more effective QCC-targeting therapeutic agents may mitigate the consequences of QCCs.

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“…For analysis of the cell cycle, cells stained with antibodies to Ki-67 were briefly exposed to Hoechst 33342 before analysis. Analysis of BrdU incorporation in LSK cells was performed as previously described (Matatall et al, 2018).…”

Section: Lead Contactmentioning

confidence: 99%